Enhanced mucosal mitochondrial function corrects dysbiosis and OXPHOS metabolism in IBD.

Background: Mitochondrial (Mito) dysfunction in IBD reduces mucosal O2 consumption and increases O2 delivery to the microbiome. Increased enteric O2 promotes blooms of facultative anaerobes (eg. Proteobacteria ) and restricts obligate anaerobes (eg. Firmicutes ). Dysbiotic metabolites negatively affect host metabolism and immunity. Our novel compound (AuPhos) upregulates intestinal epithelial cell (IEC) mito function, attenuates colitis and corrects dysbiosis in humanized Il10-/- mice. We posit that AuPhos corrects IBD-associated dysbiotic metabolism. Methods: Primary effect of AuPhos on mucosal Mito respiration and healing process was studied in ex vivo treated human colonic biopsies and piroxicam-accelerated (Px) Il10-/- mice. Secondary effect on microbiome was tested in DSS-colitis WT B6 and germ-free 129.SvEv WT or Il10-/- mice reconstituted with human IBD stool (Hu- Il10-/- ). Mice were treated orally with AuPhos (10- or 25- mg/kg; q3d) or vehicle, stool samples collected for fecal lipocalin-2 (f-LCN2) assay and microbiome analyses using 16S rRNA sequencing. AuPhos effect on microbial metabolites was determined using untargeted global metabolomics. AuPhos-induced hypoxia in IECs was assessed by Hypoxyprobe-1 staining in sections from pimonidazole HCl-infused DSS-mice. Effect of AuPhos on enteric oxygenation was assessed by E. coli Nissle 1917 WT (aerobic respiration-proficient) and cytochrome oxidase (cydA) mutant (aerobic respiration-deficient). Results: Metagenomic (16S) analysis revealed AuPhos reduced relative abundances of Proteobacteria and increased blooms of Firmicutes in uninflamed B6 WT, DSS-colitis, Hu-WT and Hu- Il10-/- mice. AuPhos also increased hypoxyprobe-1 staining in surface IECs suggesting enhanced O2 utilization. AuPhos-induced anaerobiosis was confirmed by a significant increase in cydA mutant compared to WT (O2-utlizing) E.coli . Ex vivo treatment of human biopsies with AuPhos showed significant increase in Mito mass, and complexes I and IV. Further, gene expression analysis of AuPhos-treated biopsies showed increase in stem cell markers (Lgr4, Lgr5, Lrig1), with concomitant decreases in pro-inflammatory markers (IL1ß,MCP1, RankL). Histological investigation of AuPhos-fed Px- Il10-/- mice showed significantly decreased colitis score in AuPhos-treated Px- Il10-/- mice, with decrease in mRNA of pro-inflammatory cytokines and increase in Mito complexes ( ND5 , ATP6 ). AuPhos significantly altered microbial metabolites associated with SCFA synthesis, FAO, TCA cycle, tryptophan and polyamine biosynthesis pathways. AuPhos increased pyruvate, 4-hydroxybutyrate, 2-hydroxyglutarate and succinate, suggesting an upregulation of pyruvate and glutarate pathways of butyrate production. AuPhos reduced IBD-associated primary bile acids (BA) with concomitant increase in secondary BA (SBA). AuPhos treatment significantly decreased acylcarnitines and increased L-carnitine reflective of enhanced FAO. AuPhos increases TCA cycle intermediates and creatine, energy reservoir substrates indicating enhanced OxPHOS. Besides, AuPhos also upregulates tryptophan metabolism, decreases Kynurenine and its derivatives, and increases polyamine biosynthesis pathway (Putresceine and Spermine). Conclusion: These findings indicate that AuPhos-enhanced IEC mitochondrial function reduces enteric O2 delivery, which corrects disease-associated metabolomics by restoring short-chain fatty acids, SBA, AA and IEC energy metabolism.

High fat intake sustains sorbitol intolerance after antibiotic-mediated Clostridia depletion from the gut microbiota.

Carbohydrate intolerance, commonly linked to the consumption of lactose, fructose, or sorbitol, affects up to 30% of the population in high-income countries. Although sorbitol intolerance is attributed to malabsorption, the underlying mechanism remains unresolved. Here, we show that a history of antibiotic exposure combined with high fat intake triggered long-lasting sorbitol intolerance in mice by reducing Clostridia abundance, which impaired microbial sorbitol catabolism. The restoration of sorbitol catabolism by inoculation with probiotic Escherichia coli protected mice against sorbitol intolerance but did not restore Clostridia abundance. Inoculation with the butyrate producer Anaerostipes caccae restored a normal Clostridia abundance, which protected mice against sorbitol-induced diarrhea even when the probiotic was cleared. Butyrate restored Clostridia abundance by stimulating epithelial peroxisome proliferator-activated receptor-gamma (PPAR-γ) signaling to restore epithelial hypoxia in the colon. Collectively, these mechanistic insights identify microbial sorbitol catabolism as a potential target for approaches for the diagnosis, treatment, and prevention of sorbitol intolerance.

No room at the table.

The gut microbiota prevents infection by crowding out pathogens.

Gut dysbiosis: Ecological causes and causative effects on human disease.

The gut microbiota plays a role in many human diseases, but high-throughput sequence analysis does not provide a straightforward path for defining healthy microbial communities. Therefore, understanding mechanisms that drive compositional changes during disease (gut dysbiosis) continues to be a central goal in microbiome research. Insights from the microbial pathogenesis field show that an ecological cause for gut dysbiosis is an increased availability of host-derived respiratory electron acceptors, which are dominant drivers of microbial community composition. Similar changes in the host environment also drive gut dysbiosis in several chronic human illnesses, and a better understanding of the underlying mechanisms informs approaches to causatively link compositional changes in the gut microbiota to an exacerbation of symptoms. The emerging picture suggests that homeostasis is maintained by host functions that control the availability of resources governing microbial growth. Defining dysbiosis as a weakening of these host functions directs attention to the underlying cause and identifies potential targets for therapeutic intervention.

5-ASA can functionally replace Clostridia to prevent a post-antibiotic bloom of Candida albicans by maintaining epithelial hypoxia.

Antibiotic prophylaxis sets the stage for an intestinal bloom of Candida albicans , which can progress to invasive candidiasis in patients with hematologic malignancies. Commensal bacteria can reestablish microbiota-mediated colonization resistance after completion of antibiotic therapy, but they cannot engraft during antibiotic prophylaxis. Here we use a mouse model to provide a proof of concept for an alternative approach, which replaces commensal bacteria functionally with drugs to restore colonization resistance against C. albicans . Streptomycin treatment, which depletes Clostridia from the gut microbiota, disrupted colonization resistance against C. albicans and increased epithelial oxygenation in the large intestine. Inoculating mice with a defined community of commensal Clostridia species reestablished colonization resistance and restored epithelial hypoxia. Notably, these functions of commensal Clostridia species could be replaced functionally with the drug 5-aminosalicylic acid (5-ASA), which activates mitochondrial oxygen consumption in the epithelium of the large intestine. When streptomycin-treated mice received 5-ASA, the drug reestablished colonization resistance against C. albicans and restored physiological hypoxia in the epithelium of the large intestine. We conclude that 5-ASA treatment is a non-biotic intervention that restores colonization resistance against C. albicans without requiring the administration of live bacteria.

Vi polysaccharide and conjugated vaccines afford similar early, IgM or IgG-independent control of infection but boosting with conjugated Vi vaccines sustains the efficacy of immune responses.

Introduction: Vaccination with Vi capsular polysaccharide (Vi-PS) or protein-Vi typhoid conjugate vaccine (TCV) can protect adults against Salmonella Typhi infections. TCVs offer better protection than Vi-PS in infants and may offer better protection in adults. Potential reasons for why TCV may be superior in adults are not fully understood. Methods and results: Here, we immunized wild-type (WT) mice and mice deficient in IgG or IgM with Vi-PS or TCVs (Vi conjugated to tetanus toxoid or CRM197) for up to seven months, with and without subsequent challenge with Vi-expressing Salmonella Typhimurium. Unexpectedly, IgM or IgG alone were similarly able to reduce bacterial burdens in tissues, and this was observed in response to conjugated or unconjugated Vi vaccines and was independent of antibody being of high affinity. Only in the longer-term after immunization (>5 months) were differences observed in tissue bacterial burdens of mice immunized with Vi-PS or TCV. These differences related to the maintenance of antibody responses at higher levels in mice boosted with TCV, with the rate of fall in IgG titres induced to Vi-PS being greater than for TCV. Discussion: Therefore, Vi-specific IgM or IgG are independently capable of protecting from infection and any superior protection from vaccination with TCV in adults may relate to responses being able to persist better rather than from differences in the antibody isotypes induced. These findings suggest that enhancing our understanding of how responses to vaccines are maintained may inform on how to maximize protection afforded by conjugate vaccines against encapsulated pathogens such as S. Typhi.

Bile acids as modulators of gut microbiota composition and function.

Changes in the composition of gut-associated microbial communities are associated with many human illnesses, but the factors driving dysbiosis remain incompletely understood. One factor governing the microbiota composition in the gut is bile. Bile acids shape the microbiota composition through their antimicrobial activity and by activating host signaling pathways that maintain gut homeostasis. Although bile acids are host-derived, their functions are integrally linked to bacterial metabolism, which shapes the composition of the intestinal bile acid pool. Conditions that change the size or composition of the bile acid pool can trigger alterations in the microbiota composition that exacerbate inflammation or favor infection with opportunistic pathogens. Therefore, manipulating the composition or size of the bile acid pool might be a promising strategy to remediate dysbiosis.

Predicting the Next Superspreader.

The spread of multidrug-resistant zoonotic pathogens, such as Salmonella, within livestock is of concern for food safety. The spread of Salmonella on the farm is escalated by superspreaders, which shed the pathogen at high numbers with their feces. However, there are currently no biomarkers to identify potential superspreaders. Kempf and coworkers determined that a potent early inflammatory response to Salmonella infection and changes in the microbiota composition are associated with the superspreader phenotype in pigs (F. Kempf, G. Cordoni, A.M. Chaussé, R. Drumo, et al., mSystems, in press, https://doi.org/10.1128/msystems.00852-22). Since these biomarkers only develop during Salmonella infection, additional work is needed to predict animals that have the potential to become superspreaders.

Identification of collaborative cross mouse strains permissive to Salmonella enterica serovar Typhi infection.

Salmonella enterica serovar Typhi is the causative agent of typhoid fever restricted to humans and does not replicate in commonly used inbred mice. Genetic variation in humans is far greater and more complex than that in a single inbred strain of mice. The Collaborative Cross (CC) is a large panel of recombinant inbred strains which has a wider range of genetic diversity than laboratory inbred mouse strains. We found that the CC003/Unc and CC053/Unc strains are permissive to intraperitoneal but not oral route of S. Typhi infection and show histopathological changes characteristic of human typhoid. These CC strains are immunocompetent, and immunization induces antigen-specific responses that can kill S. Typhi in vitro and control S. Typhi in vivo. Our results indicate that CC003/Unc and CC053/Unc strains can help identify the genetic basis for typhoid susceptibility, S. Typhi virulence mechanism(s) in vivo, and serve as a preclinical mammalian model system to identify effective vaccines and therapeutics strategies.

The IRE1α-XBP1 Signaling Axis Promotes Glycolytic Reprogramming in Response to Inflammatory Stimuli.

Immune cells must be able to adjust their metabolic programs to effectively carry out their effector functions. Here, we show that the endoplasmic reticulum (ER) stress sensor Inositol-requiring enzyme 1 alpha (IRE1α) and its downstream transcription factor X box binding protein 1 (XBP1) enhance the upregulation of glycolysis in classically activated macrophages (CAMs). The IRE1α-XBP1 signaling axis supports this glycolytic switch in macrophages when activated by lipopolysaccharide (LPS) stimulation or infection with the intracellular bacterial pathogen Brucella abortus. Importantly, these different inflammatory stimuli have distinct mechanisms of IRE1α activation; while Toll-like receptor 4 (TLR4) supports glycolysis under both conditions, TLR4 is required for activation of IRE1α in response to LPS treatment but not B. abortus infection. Though IRE1α and XBP1 are necessary for maximal induction of glycolysis in CAMs, activation of this pathway is not sufficient to increase the glycolytic rate of macrophages, indicating that the cellular context in which this pathway is activated ultimately dictates the cell's metabolic response and that IRE1α activation may be a way to fine-tune metabolic reprogramming. IMPORTANCE The immune system must be able to tailor its response to different types of pathogens in order to eliminate them and protect the host. When confronted with bacterial pathogens, macrophages, frontline defenders in the immune system, switch to a glycolysis-driven metabolism to carry out their antibacterial functions. Here, we show that IRE1α, a sensor of ER stress, and its downstream transcription factor XBP1 support glycolysis in macrophages during infection with Brucella abortus or challenge with Salmonella LPS. Interestingly, these stimuli activate IRE1α by independent mechanisms. While the IRE1α-XBP1 signaling axis promotes the glycolytic switch, activation of this pathway is not sufficient to increase glycolysis in macrophages. This study furthers our understanding of the pathways that drive macrophage immunometabolism and highlights a new role for IRE1α and XBP1 in innate immunity.

The Lack of Natural IgM Increases Susceptibility and Impairs Anti-Vi Polysaccharide IgG Responses in a Mouse Model of Typhoid.

Circulating IgM present in the body prior to any apparent Ag exposure is referred to as natural IgM. Natural IgM provides protective immunity against a variety of pathogens. Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of typhoid fever in humans. Because mice are not permissive to S. Typhi infection, we employed a murine model of typhoid using S. enterica serovar Typhimurium expressing the Vi polysaccharide (ViPS) of S. Typhi (S. Typhimurium strain RC60) to evaluate the role of natural IgM in pathogenesis. We found that natural mouse IgM binds to S. Typhi and S. Typhimurium. The severity of S. Typhimurium infection in mice is dependent on presence of the natural resistance-associated macrophage protein 1 (Nramp1) allele; therefore, we infected mice deficient in secreted form of IgM (sIgM) on either a Nramp1-resistant (129S) or -susceptible (C57BL/6J) background. We found that the lack of natural IgM results in a significantly increased susceptibility and an exaggerated liver pathology regardless of the route of infection or the Nramp1 allele. Reconstitution of sIgM-/- mice with normal mouse serum or purified polyclonal IgM restored the resistance to that of sIgM+/+ mice. Furthermore, immunization of sIgM-/- mice with heat-killed S. Typhi induced a significantly reduced anti-ViPS IgG and complement-dependent bactericidal activity against S. Typhi in vitro, compared with that of sIgM+/+ mice. These findings indicate that natural IgM is an important factor in reducing the typhoid severity and inducing an optimal anti-ViPS IgG response to vaccination.

The Vi Capsular Polysaccharide of Salmonella Typhi Promotes Macrophage Phagocytosis by Binding the Human C-Type Lectin DC-SIGN.

Capsular polysaccharides are common virulence factors of extracellular, but not intracellular bacterial pathogens, due to the antiphagocytic properties of these surface structures. It is therefore paradoxical that Salmonella enterica subspecies enterica serovar Typhi, an intracellular pathogen, synthesizes a virulence-associated (Vi) capsule, which exhibits antiphagocytic properties. Here, we show that the Vi capsular polysaccharide has different functions when S. Typhi interacts with distinct subsets of host phagocytes. The Vi capsular polysaccharide allowed S. Typhi to selectively evade phagocytosis by human neutrophils while promoting human macrophage phagocytosis. A screen of C-type lectin receptors identified human DC-SIGN as the receptor involved in macrophage binding and phagocytosis of capsulated S. Typhi. Consistent with the anti-inflammatory activity of DC-SIGN, purified Vi capsular polysaccharide reduced inflammatory responses in macrophages. These data suggest that binding of the human C-type lectin receptor DC-SIGN by the Vi capsular polysaccharide contributes to the pathogenesis of typhoid fever. IMPORTANCE Salmonella enterica subspecies enterica serovar Typhi is the causative agent of typhoid fever. The recent emergence of S. Typhi strains which are resistant to antibiotic therapy highlights the importance of vaccination in managing typhoid fever. The virulence-associated (Vi) capsular polysaccharide is an effective vaccine against typhoid fever, but the role the capsule plays during pathogenesis remains incompletely understood. Here, we identify the human C-type lectin receptor DC-SIGN as the receptor for the Vi capsular polysaccharide. Binding of capsulated S. Typhi to DC-SIGN resulted in phagocytosis of the pathogen by macrophages and induction of an anti-inflammatory cytokine response. Thus, the interaction of the Vi capsular polysaccharide with human DC-SIGN contributes to the pathogenesis of typhoid fever and should be further investigated in the context of vaccine development.

The microbiome and gut homeostasis.

Changes in the composition of the gut microbiota are associated with many human diseases. So far, however, we have failed to define homeostasis or dysbiosis by the presence or absence of specific microbial species. The composition and function of the adult gut microbiota is governed by diet and host factors that regulate and direct microbial growth. The host delivers oxygen and nitrate to the lumen of the small intestine, which selects for bacteria that use respiration for energy production. In the colon, by contrast, the host limits the availability of oxygen and nitrate, which results in a bacterial community that specializes in fermentation for growth. Although diet influences microbiota composition, a poor diet weakens host control mechanisms that regulate the microbiota. Hence, quantifying host parameters that control microbial growth could help define homeostasis or dysbiosis and could offer alternative strategies to remediate dysbiosis.

Host cells subdivide nutrient niches into discrete biogeographical microhabitats for gut microbes.

Changes in the microbiota composition are associated with many human diseases, but factors that govern strain abundance remain poorly defined. We show that a commensal Escherichia coli strain and a pathogenic Salmonella enterica serovar Typhimurium isolate both utilize nitrate for intestinal growth, but each accesses this resource in a distinct biogeographical niche. Commensal E. coli utilizes epithelial-derived nitrate, whereas nitrate in the niche occupied by S. Typhimurium is derived from phagocytic infiltrates. Surprisingly, avirulent S. Typhimurium was shown to be unable to utilize epithelial-derived nitrate because its chemotaxis receptors McpB and McpC exclude the pathogen from the niche occupied by E. coli. In contrast, E. coli invades the niche constructed by S. Typhimurium virulence factors and confers colonization resistance by competing for nitrate. Thus, nutrient niches are not defined solely by critical resources, but they can be further subdivided biogeographically within the host into distinct microhabitats, thereby generating new niche opportunities for distinct bacterial species.

To breathe or not to breathe?

Listeria monocytogenes uses respiration to sustain a risky fermentative lifestyle during infection.

Assessment of Murine Colon Inflammation Using Intraluminal Fluorescence Lifetime Imaging.

Inflammatory bowel disease (IBD) is typically diagnosed by exclusion years after its onset. Current diagnostic methods are indirect, destructive, or target overt disease. Screening strategies that can detect low-grade inflammation in the colon would improve patient prognosis and alleviate associated healthcare costs. Here, we test the feasibility of fluorescence lifetime imaging (FLIm) to detect inflammation from thick tissue in a non-destructive and label-free approach based on tissue autofluorescence. A pulse sampling FLIm instrument with 355 nm excitation was coupled to a rotating side-viewing endoscopic probe for high speed (10 mm/s) intraluminal imaging of the entire mucosal surface (50-80 mm) of freshly excised mice colons. Current results demonstrate that tissue autofluorescence lifetime was sensitive to the colon anatomy and the colonocyte layer. Moreover, mice under DSS-induced colitis and 5-ASA treatments showed changes in lifetime values that were qualitatively related to inflammatory markers consistent with alterations in epithelial bioenergetics (switch between ß-oxidation and aerobic glycolysis) and physical structure (colon length). This study demonstrates the ability of intraluminal FLIm to image mucosal lifetime changes in response to inflammatory treatments and supports the development of FLIm as an in vivo imaging technique for monitoring the onset, progression, and treatment of inflammatory diseases.

Virulence factors perforate the pathogen-containing vacuole to signal efferocytosis.

Intracellular pathogens commonly reside within macrophages to find shelter from humoral defenses, but host cell death can expose them to the extracellular milieu. We find intracellular pathogens solve this dilemma by using virulence factors to generate a complement-dependent find-me signal that initiates uptake by a new phagocyte through efferocytosis. During macrophage death, Salmonella uses a type III secretion system to perforate the membrane of the pathogen-containing vacuole (PCV), thereby triggering complement deposition on bacteria entrapped in pore-induced intracellular traps (PITs). In turn, complement activation signals neutrophil efferocytosis, a process that shelters intracellular bacteria from the respiratory burst. Similarly, Brucella employs its type IV secretion system to perforate the PCV membrane, which induces complement deposition on bacteria entrapped in PITs. Collectively, this work identifies virulence factor-induced perforation of the PCV as a strategy of intracellular pathogens to generate a find-me signal for efferocytosis.

Identification of a gut microbiota member that ameliorates DSS-induced colitis in intestinal barrier enhanced Dusp6-deficient mice.

Strengthening the gut epithelial barrier is a potential strategy for management of gut microbiota-associated illnesses. Here, we demonstrate that dual-specificity phosphatase 6 (Dusp6) knockout enhances baseline colon barrier integrity and ameliorates dextran sulfate sodium (DSS)-induced colonic injury. DUSP6 mutation in Caco-2 cells enhances the epithelial feature and increases mitochondrial oxygen consumption, accompanied by altered glucose metabolism and decreased glycolysis. We find that Dusp6-knockout mice are more resistant to DSS-induced dysbiosis, and the cohousing and fecal microbiota transplantation experiments show that the gut/fecal microbiota derived from Dusp6-knockout mice also confers protection against colitis. Further culturomics and mono-colonialization experiments show that one gut microbiota member in the genus Duncaniella confers host protection from DSS-induced injury. We identify Dusp6 deficiency as beneficial for shaping the gut microbiota eubiosis necessary to protect against gut barrier-related diseases.

The metabolic footprint of Clostridia and Erysipelotrichia reveals their role in depleting sugar alcohols in the cecum.

BACKGROUND: The catabolic activity of the microbiota contributes to health by aiding in nutrition, immune education, and niche protection against pathogens. However, the nutrients consumed by common taxa within the gut microbiota remain incompletely understood. METHODS: Here we combined microbiota profiling with an un-targeted metabolomics approach to determine whether depletion of small metabolites in the cecum of mice correlated with the presence of specific bacterial taxa. Causality was investigated by engrafting germ-free or antibiotic-treated mice with complex or defined microbial communities. RESULTS: We noted that a depletion of Clostridia and Erysipelotrichia from the gut microbiota triggered by antibiotic treatment was associated with an increase in the cecal concentration of sugar acids and sugar alcohols (polyols). Notably, when we inoculated germ-free mice with a defined microbial community of 14 Clostridia and 3 Erysipelotrichia isolates, we observed the inverse, with a marked decrease in the concentrations of sugar acids and polyols in cecal contents. The carbohydrate footprint produced by the defined microbial community was similar to that observed in gnotobiotic mice receiving a cecal microbiota transplant from conventional mice. Supplementation with sorbitol, a polyol used as artificial sweetener, increased cecal sorbitol concentrations in antibiotic-treated mice, which was abrogated after inoculation with a Clostridia isolate able to grow on sorbitol in vitro. CONCLUSIONS: We conclude that consumption of sugar alcohols by Clostridia and Erysipelotrichia species depletes these metabolites from the intestinal lumen during homeostasis. Video abstract.

The longitudinal and cross-sectional heterogeneity of the intestinal microbiota.

A central goal of microbiome research is to understand the factors that balance gut-associated microbial communities, thereby creating longitudinal and cross-sectional heterogeneity in their composition and density. Whereas the diet dictates taxa dominance, microbial communities are linked intimately to host physiology through digestive and absorptive functions that generate longitudinal heterogeneity in nutrient availability. Additionally, the host differentially controls the access to electron acceptors along the longitudinal axis of the intestine to drive the development of microbial communities that are dominated by facultatively anaerobic bacteria in the small intestine or obligately anaerobic bacteria in the large intestine. By secreting mucus and antimicrobials, the host further constructs microhabitats that generate cross-sectional heterogeneity in the colonic microbiota composition. Here we will review how understanding the host factors involved in generating longitudinal and cross-sectional microbiota heterogeneity helps define physiological states that are characteristic of or appropriate to a homeostatic microbiome.